Birth control method involving monitoring of axillary androstenol and dehydroepiandrosterone

ABSTRACT

A novel method of determining the onset of the fertile period of a human female is disclosed comprising the step of monitoring the axillary secretions of said female during the course of her menstrual cycle to determine variations in the concentration of androstenol or dehydroepiandrosterone sulfate. A first significant increase in the concentration of either of those compounds following the cessation of menses is indicative of the onset of the fertile period. In each case, a follicular phase peak occurs which exceeds by at least twice the concentration of those compounds present at any other time during the menstrual cycle. As a result, the monitoring of these compounds may act as reliable indicators predicting the occurrence of ovulation in five to seven days, and/or indicating contraceptive measures can still be taken to avoid pregnancy or sexual intercourse can promote fertility.

CROSS-REFERENCE TO RELATED PATENTS AND APPLICATIONS

This is a division of application Ser. No. 695,053, filed Jan. 25, 1985,now U.S. Pat. No. 4,670,401, issued June 2, 1987.

The present application is related to U.S. Pat. Nos. 3,986,494 entitled"Method for Predicting and Detecting Ovulation" (Preti and Huggins);4,010,738, entitled "Method of Predicting and Detecting Ovulation"(Preti and Huggins); 4,119,089, entitled "Method of Predicting andDetermining Ovulation by Monitoring the Concentration of VolatileSulfur-Containing Compounds Present in Mouth Air" (Preti, Huggins andTonzetich); and 4,385,125, entitled "Method Detecting Ovulation byMonitoring Dodecanol Concentration in Saliva" (Preti, Kostelc, Tonzetichand Higgins), each of which patents is hereby incorporated by referenceas if fully set forth herein.

This application is further related to U.S. application No. 694,947,filed Jan. 25, 1985, entitled "The Use of Male Essence, IncludingAndrostenol and Dehydroepiandrosterone Sulfate to Treat Luteal PhaseDefects and Failure to Ovulate in Human Females", which application isalso incorporated by reference as if fully set forth herein.

This application is also related to U.S. application No. 594,946, filedJan. 25, 1985, entitled "Method Using Pooled Female Axillary EssenceFrom Multiple Donors to Alter Menstrual Cycle Timing", which applicationis hereby incorporated by reference as if fully set forth herein.

BACKGROUND OF THE INVENTION

The present invention relates to the field of reproductive biology, andmore particularly to methods used by reproductive biologists,gynecologists and/or obstetricians to predict in human females theprobability that a given menstrual cycle type is presumptively fertileor infertile, the timing of the menstrual cycle, and/or the onset of thefertile period, the time of ovulation. The field of the presentinvention includes the field of detection and diagnosis of ovulation inhuman females through the detection of secondary characteristicsoccurring during or at the time of ovulation, and more particularly, tothe identification and detection of such secondary characteristics asthey appear in human females.

For many years there has been a need to detect and diagnose the precisetime of ovulation in a given female mammal. It can be of greatimportance, for example, to pinpoint the time of ovulation to ensurethat fertilization occurs or to prevent conception. Alternatively, itmay be important for other medical reasons to diagnose ovulation.

To some extent, methods of diagnosing ovulation are disclosed in theabove-identified related patents. Additionally, the occurrence ofovulation can be established with some certainty through various priorart methods. For a review of many of the known surgical, clinical,biochemical or hystological techniques for diagnosing ovulation, pleaserefer to the descriptions appearing in the above-mentioned relatedpatents and patent applications, particularly to columns 1-5 of U.S.Pat. No. 4,119,089.

Perhaps the most popular and widely used method of estimating the timeof ovulation relies upon the graphic recording of the waking temperatureat basal conditions (hereinafter referred to as the BBT method). Usingthis method a dedicated woman with uniform daily habits can determinethe time of ovulation within two days after its occurrence. In recordingthe basal body temperature, a rise in temperature is commonly associatedwith the beginning of the luteal phase, but can vary from the actualtime of ovulation by as much as 72 hours. FIG. 1 of U.S. Pat. No.4,119,089 illustrates a theoretical basal body temperature chart showinga biphasic cycle having a lowered body temperature during the follicularphase, and a sustained raised body temperature during the luteal phase.

Another technique which is commonly used to determine the time ofovulation in human females in the charting of concentrations of certainhormones appearing in the blood. In humans, a preovulatory rise in serumestrogens coupled with a sharp rise in luteinizing hormone (LH) levelsas determined by radio-immunoassay of serially drawn blood samples, isperhaps the most accurate indicator of impending ovulation. Ovulationmost likely occurs 12-24 hours after maximum LH levels. A subsequentpersistent increase in levels of serum progesterone indicates thatovulation has occurred. Since these determinations are expensive and notwidely available, clinical parameters such as BBT parameters are mostoften used to determine the time of ovulation.

The field of the present invention also relates to methods of predictingthe onset of the fertile period. It is generally accepted that themaximum survival function of spermatozoa capable of fertilizing an ovumis approximately three days following coitus. Although theoretically anycoitus prior to ovulation entails a certain risk of pregnancy, as apractical matter, abstinence from unprotected sexual intercourse for atleast 3 days (preferably up to 5 days) prior to ovulation is generallyconsidered sufficient to avoid pregnancy. It is generally recognizedthat a human ovum is (in vivo) fertilizable for about 12 hours andcertainly for no more than 1 day following ovulation. The human fertileperiod, then, is made up of no more than 4 to 6 days out of the entiremenstrual cycle. If it were possible to accurately predict this fertileperiod, in order to avoid pregnancy it would only be necessary toabstain from unprotected intercourse or use alternate birth controlmethods during that 4-6 day "fertile period" rather than for the entiremenstrual cycle.

Notwithstanding the methods of the aforementioned related patents, theonly technique yet widely used for "predicting" the fertile period of afemale is the method which relies upon basal body temperaturedetermination of ovulation in a plurality of preceding cycles todetermine the expected time of ovulation for future cycles. This methodis not really directed to ascertaining the precise fertile period for agiven cycle, but rather is intended to identify a period during whichcoitus is statistically likely to produce pregnancy. Based on calendarrecords of 21,499 menstrual cycles experienced by 592 healthy womenliving in Switzerland, 9.75 of all menstrual cycles range in length from6-23 days, 80.1% into the range from 24-34 days, and another 10.2% coverthe remainder of menstrual cycles from 35 to 409 days in length. Themodal cycle length of the cycle of 28 days duration and the averagecycle length amounts to 29.2 days. See "The Degree of Variability in theLength of the Menstrual Cycle in Correlation with Age of Woman", by R.F. Vollman, Gynaecologia, 142(5):310-314 (November 1956). Since thisinformation is based upon past performance, and since the time ofovulation varies markedly between different individuals as well betweencycles of a given individual, the period for abstinence must be longenough to considerably reduce the possibility of pregnancy. As explainedin U.S. Pat. No. 4,119,089, taking into account the variability inmenstrual cycle length, even the most regular women probably need toabstain from unprotected coitus for more than one-third of the totalmenstrual cycle in order to avoid pregnancy. Accordingly, a long feltneed exists for methods of more accurately predicting or detecting theonset of the fertile period for a given cycle, such that coitus can beavoided, or other contraceptive methods employed, during the fertileperiod of that cycle.

The present invention also relates to the field of mammalian chemicalcommunication, and more particularly to research involving mammalianpheromones. It has long been known that the estrus cycles of certainmammalian species are effected by pheromones and that their estruscycles may be artificially manipulated. For examples, estrus cycles ofrats can be manipulated through the use of odors collected form femalesduring specific phases of the estrous cycle. See McClintock, M.K.,"Pheromonal Regulation of the Ovarian Cycle: Enhancement, Suppressionand Synchrony", Pheromones and Reproduction in Mammals, edited by J. G.Vanderbergh. New York: Academic Press, pp. 113-149 (1983) andMcClintock, M. K., "Estrous Synchrony: Modulation of Ovarian CycleLength by Female Pheromones"Physiol. Behav. 32:701-705 (1984). Inaddition, preovulatory cervical mucus mixed with water and sprayed intothe noses of a group of female Holstein cows advanced and synchronizedthe time of estrous. See Izard, M. K., "Pheromones and Reproduction inDomestic Animals"Pheromones and Reproduction in Mammals, edited by J. G.Vandenbergh, New York: Academic Press, pp. 253-281 (1983) and Izard, M.K. and Vanderbergh, J. G., "Priming Pheromones From Oestrous CowsIncrease Synchronization of Oestrus in Dairy Heifers After PGF-2Injection". J. Reprod. Fert. 66:189-196, (1982). Notwithstanding theseand other reports of elaborate pheromonal systems in other mammals therehas been no experimental evidence that pheromonal systems operate inhumans.

More recently a number of studies have suggested that possibility thathuman odors act in a manner analogous to primer pheromones in animalsand alter reproductive endocrinology. Although anecdotal reports ofaphrodesiacs are as old as modern civilization, the possibility thathuman reproductive biology can be altered by pheromones was notconsidered seriously until McClintock published a report in 1971 thatmenstrual synchrony occurred among certain women attending apredominantly female university. In "Menstrual Synchrony andSuppression", Nature, 229:244-245 (Jan. 22, 1971) McClintock reportedthat "social interaction" can have a strong effect on the menstrualcycle of women living together in a college dormitory. The McClintockstudy compared the dates of menstrual onset for roommates and closefriends, and for living groups. McClintock reported that a significantincrease in menstrual synchrony was found among roommates, among closestfriends, and among roommates and closest friends combined. McClintockhypothesized that the synchrony could be due to some factor other thantime spent with an individual such as the available food and/orsynchrony of the moon. McClintock further speculated that synchronymight parallel the Whitten effect in mice in which suppression ofoestrous in groups of females may be released by the introduction of amale mouse pheromone. McClintock suggested that synchrony might resultfrom a pheromonal interaction of suppression among close friend groups,followed by a periodic release due to the presence of males on theweekend. McClintock stated:

"However, this would be insufficient to explain the synchrony whichoccurred among roommates and close friends but did not occur throughoutthe dormitory."

A further possible explanation advanced by McClintock was the awarenessof menstrual cycles among friends, however a sample taken from thedormitory indicated that 47% were not conscious of their friends'menstrual cycles, and of the 53% who were, 48% were only vaguely aware.While McClintock was able to conclude that a significant factor insynchrony is that individuals in the group spend time together,McClintock was ultimately unable to state whether the mechanismunderlying this phenomenon is pheromonal, mediated by awareness, or someother process, indicating that the question "still remains open forspeculation and investigation". McClintock also explained:

"Exposure to males may not be the significant factor". It may be, forexample, that those with longer cycles are less likely to spend timewith males. However, many subjects spontaneously indicated that theybecame more regular and had shorter cycles when they dated more often.For example, one subject reported that she had a cycle length of sixmonths until she began seeing males more frequently. Her cycle lengththen shortened to 4.5 weeks. Then, when she stopped seeing males asoften, her cycle lengthened again. Whether this is due to a pheromonemechanism similar to the Lee-Boot effect in mice has yet to bedetermined."[Lee and Boot, Acta physiol. Pharamacol. Neerl., 5, 213(1956)].

Subsequent investigators have also considered the effect on menstrualsynchrony of females in different living conditions. In "MenstrualSynchrony in Female Undergraduates Living on a Coeducational Campus",Psychoneuroendocrinology, 5:245-252 (1980), Graham and McGrew reportinvestigations of menstrual synchrony among female undergraduates livingon a coeducational university campus. A significant trend towardsynchrony was found for closest friends, but no significant effectoccurred for neighbors or randomized pairs. Nor did any significantcorrelation emerge between cycle length, duration, duration ofmenstruation, or the amount and nature of social interaction with males.Graham and McGrew accordingly conclude that the amount of timeindividuals spend together, not similar living conditions, is thesignificant fact in synchrony. Graham and McGrew further conclude thatthe "how and why" of menstrual synchrony remain unknown, indicatingthat, as suggested by Rogel in "A Critical Evaluation of the Possibilityof Higher Primate Reproductive and Sexual Pheromones", Psychol. Bull.85:810-830 (1978), menstrual synchrony cannot be explained by anyexisting hypothesis other than a pheromonal one.

In 1981, Quadagno et al reported a tendency toward menstrual synchronywhich was greatest between roommates and close friends. See Quadagno,D.M., Shubeita, H.E., Deck J. and Francoeur, D., "Influence of MaleSocial Contacts, Exercise and All Female Living Conditions on theMenstrual Cycle". Psychoneuroendorcrinol. 6:239-244 91981). Casualexercise performed regularly was associated with longer menstrual cycleswhereas spending "social time" with a male was reported not to have anyeffect on the length of the menstrual cycle for the women in the study.Noting McClintock's finding that "women seeing males less than 3 times aweek experience significantly longer cycles than women who spent timewith males more than 3 times per week", Quadagno nonetheless confirmsGraham and McGrew's findings that no significant correlation existsbetween cycle length and the amount and nature of social interactionwith males.

Most recently, Veith et al have reported that exposure to men has thecapacity to shorten the menstrual cycle in women. See "Exposure to MenInfluence the Occurrence of Ovulation in Woman", Physiology and Behavior31:313-315 (1983). Veith et al report that women who spent two or morenights with men during a 40 day period exhibited a significantly higherrate of ovulation as determined by basal body temperature charts thanthose spending no or one nights. Cycle length was not effected bysleeping arrangements, and the frequency of sexual intercourse was saidto be unrelated to either cycle length or likelihood of ovulation. Themechanism underlying this phenomenon was reported to be "unknown", butwas "conjectured" to be pheromonal in nature. Veith et al thus concludethat "a significant variable contributing to the likelihood of ovulationare the number of nights a woman spends in the same bed with a man".Veith et al further conclude that cycle length "was not influenced bythis factor", noting that the "lack of replication of the earlier[McClintock] study concerning the lengthening of cycles in those womenwith limited exposure to males is surprising". Veith suggests that thenaturalistic conditions resulting from subjects being enrolled in acoeducational institution may provide participants with sufficientexposure to men so that their cycle length would not be undulyincreased.

A number of studies have investigated the possible effect of underarmperspiration on menstrual synchrony. In 1977, Russell, Switz andThompson reported on the effects of using the female axillary sweat of asingle, so-called "driver" female as a stimulus toward menstrualsynchrony. See "Olfactory Influences on the Human Menstrual Cycle"presented at the meeting of the American Association for Advancement ofScience, San Francisco, June 1977 and published in Pharmac. Biochm.Behav. 13(5):1-2 (1980). Russell et al had female volunteers rub ontheir upper lips cotton pads containing the perspiration odor of aparticular donor female. After four months the menstrual onset dates ofthe volunteers were significantly closer to that of donor female thanwere the cycles of the control group who received the application butnot the odor. The particular female donor subject had a history of avery regular menstrual cycle of 28 days and no significant history ofmenstrual problems. She had demonstrated a previous experience of"driving" another woman's menstrual cycle on three separate occasionsover three consecutive years, did not use underarm deodorant nor shaveunder her arms. These data showed that the mean difference from onset ofthe menstrual cycle of the subjects from the donor was 9.3 days in thepretreatment month and 3.4 days in the post treatment month for theexperimental group, and that this change showed statistical significanceover the control group. Russel et al thus conclude that "odors from onewoman may influence the menstrual cycle of another and that these odorscan be collected from the underarm, stored as frozen samples, for atleast short periods, and placed on another woman." The experiment wasfurther said to support the theory that odor is a communicative elementin human menstrual synchrony, and that at least a rudimentary form ofolfactory control of the hormonal system is occuring in humans in asimilar fashion to that found in other mammals.

As Russell et al noted, it is possible that volatile substances aretransferred to the nose even though the subject has no awareness ofthem. It is also possible that the mechanism of transfer in the Russellet al study did not involve the nose at all, but diffusion of chemicalcompounds through the skin which may occur when the sample was placed onthe subject's upper lip.

Underarm sweat comprises secretions of the apocrine, eccrine andsebaceous glands. Analyses of apocrine secretion have shown the presenceof protein (10%), cholesterol (1%) and two androgen steroids:andosterone sulfate and dehydroepiandrosterone sulfate (0.2%). Theapocrine secretion as collected at the skin surface is odorless;however, incubation with the resident skin bacteria results in an odorprofile unique to that organism. The micrococci bacteria give an "acidodor" to the secretion which has been characterized by head spaceanalysis as isovoleric acid. See Labows, J. N. 1979. "Human odors--whatcan they tell us!"Perfumer and Flavorist 4:12-17. The diptheroidbacteria give a similar head space profile but the observed odor is themore distinct "apocrine (human) odor" usually associated with theaxillary areas.

Similar correlations of odor quality and bacterial populations werefound in vivo. The axillary microflora of 229 subjects have beencharacterized quantitatively and their results correlated with the typeof donor found. Micrococcaceae were present in all subjects and were thedominant organism when a faint or acid odor was noted. The aerobicdiptheroids were found in 85% of the males and 66% of the females andwere associated with a pungent, apocrine odor. There were no significantdifferences related to handedness or presence of axillary hair. SeeLeyden, J. J., McGinley, K. J., Hoelzle, E., Labows, J. N. and Kligman,A. M. 1981, "The Microbiology of the Human Axillae and its Relation toAxillary Odors", J. Invest. Dermatol. 77:413-416.

The "apocrine odor" is similar to that of androst-16-en-3-one. SeeLabows, J. N. 1979, supra. Neither this steroid nor androst-16-en-3-ol,a musky odorant are present in original odorless secretion but they havebeen shown to be present in axillary sweat. See Bird, S. and Gower, D.B. 1982, "Axillary 5-androst-16-en-3-one, Cholesterol and Squalene inMen: Preliminary Evidence for 5-androst-16-en-3-one Being a Product ofBacterial Action". J. Steroid Biochm. 17:517-522; Bird, S. and Gower, D.B., "Measurement of 5-androst-16-en-one in Human Axillary Secretions byRadioimmunoassay", J. Endorcrinol. 85:80-90; and Brooksbank, B. W. L.,Brown, R. and Gustafsson, J. A. 1974, "The Detection of5-androst-16-en-3-ol in Human Male Axillary Sweat", Experientia30:864-865. In experiments in which axillary bacteria where incubatedwith sterile apocrine sweat, typical pungent malodor was produced onlywith aerobic diptheroids. This included both isovoleric acid and anothercomponent which was pungent. The odor is similar in character toandrost-16-en-3-one and this material has been detected on axillary padsusing radio-immunoassay procedures. See Bird, S. and Gower D.B. supra.Studies of extracts of odorous cultures of diptheroids grown on apocrinesecretion by GC/MS as yet have not revealed detectable levels ofandrostenone. It is reasonable to assume, however, that further studieswill reveal them since they are detected in axillary washings. SeePersonal Communication, 1984. Dr. J. N. Labows, Monell Chemical Senses,Center.

Apart from the above-mentioned data, Graham et al describe the remainingevidence for pheromonal communication of a human female's hormonalstatus as being "largely circumstantial and fragmentary". For example,Graham et al cite work on short-chain fatty acids in human vaginalsecretions; vaginal odors; odor discrimination studies; effects ofalleged human pheromonal preparations on subject performance; andscent-marking experiments in public places using androstenone, amammalian pheromone, which is said to alter spontaneous behavior. Seefor example, Clark, Klassnick and Watson, "Human Responses toScent-Marking with the Putative Pheromone Androstenone" (1978)(unpublished manuscript cited in Graham et al supra).

In addition to the above, numerous publications discuss various factorssuch as lunar synchrony which may effect the length of the humanmenstrual cycle. See "The Menstrual Cycle" by Rudolph V. Vollman, M. D.,Vol. 7, Major Problems in Obstetrics and Gynecology, edited by EmmanualA. Freidman, M. D.; Treloar, "Variation of the Human Menstrual CycleThrough Reproductive Life", International Journal of Fertility,12(1):77-126 (1967). More recently, Winnifred Berg Cutler has reportedupon the relationship between the lunar and menstrual cycle phases. See"Lunar and Menstrual Phase Locking", by Winnifred Cutler, AmericanJournal of Obstetrics and Gynecology 137:834 (1980). In this study,reference is made to data showing the mean and median of samplemenstrual cycle data to be 29.5 days, and notes the coincidence of thiscycle length and the 29.5 day lunar cycle. Cutler has noted that 98% ofthe cycles of 29.5±1 day in length are ovulatory, and has suggested thatovulation may frequently occur during the new moon part of the cycle.

In addition to the factors discussed above, sexual behavior has oftenbeen discussed as a factor which contributed to the rhythm of the humancycle and subsequent fertility. Cutler has published extensively on thepossible interrelationship between infertility and first coitus, sexualbehavior frequency and menstrual cycle length, short hyperthermic phasesand sporadic sexual behavior in woman, and relationships betweenestrogen level, hot flashes and sexual behavior in perimenopausal woman.See Cutler et al "Infertility and Age at First Coitus; A PossibleAssociation", J. Biosoc. Sci. 11: 425-432 (1979); Cutler et al, "SexualBehavior Frequency and Menstrual Cycle Length in Mature PremenopausalWomen", Psychoneuroendocrinology 4:297-309 (1979); Cutler et al, "LutealPhase Defects: A Possible Relationship Between Short Hyperthermic Phaseand Sporadic Sexual Behavior in Women", Hormones and Behavior 13:214-218(1979); Cutler et al, "Sporadic Sexual Behavior and Menstrual CycleLength in Women", Hormones and Behavior 14:163-172 (1980); Cutler et al,"The psychoneuroendocrinology of the Ovulatory Cycle of Woman, AReview", Psychoneuroendocrinology 5:1980; and Cutler et al,"Relationships Between Estrogen Level, Hot Flashes and Sexual Behaviorin perimenopausal Women", Neuroendocrinology Letters 5:185 (1983).

In addition to the Vollman and Treloar studies discussed above, whichclearly suggest that women whose cycles approach a 29 day span have thehighest likelihood of a fertile cycle, Cutler et al have demonstratedthat women who have regular weekly, heterosexual activity have menstrualcycles of about 29 days, whereas women who either have sporadic sexualactivity or who are celibate tend to have a higher frequency of aberrantlength cycles (25 or less or 34 or more days). These studies suggestthat leuteal phase defects (shortened hyperthermic phases) and sporadicsexual activity in the luteal phase are associated in a population ofinfertility patients. Delayed age of first coitus is also associatedwith a higher frequency of subsequent infertility.

Even more recently, Cutler et al have evaluated whether self stimulation(masturbation), coitus, and or heterosexual genital stimulation by a manwithout coitus, could be differentiated in terms of their relativeassociation to fertility. In this study presumptive fertility (P.fertility) was evaluated by the menstrual cycle length and by analysisof basal body temperature charts. It was found that weekly heterosexualbehavior is consistently associated with menstrual cycle lengths of29.5±3 days and that self stimulation behavior does not show a similarassociation between weekly behavior and circa 29 day menstrual cyclelengths. Women who report regular weekly sex with men are found to havea greater incidence of presumptively fertile menstrual cycles than thosewho report sporadic sexual activity. In the Cutler et al study the chiefproblem associated with less frequent sexual activity is less a failureto ovulate but rather a short luteal phase. A fertile menstrual cyclerequires both an ovulation and an adequately long luteal phase to ensuresufficient time and steroid output to prepare the endometrium forimplantation. The Cutler et al study shows that sexually active womenwho have regular, stable patterns of sexual activity have significantlymore fertile type cycles than women of the same age who have sporadicpatterns of sexual activity. The hypothesis that normalization of themenstrual cycle is due to something more than genital stimulation, butrequires the presence of a male partner, is consistent with thesuggestions of McClintock, the data of Quadagno, and numerous studiesand observations conducted with non-human primates and other mammals,some of which suggest the presence of a male or his odors are importantin regulating cyclicity and/or fertility.

The influence of mammals or their odors on the estrous cycles of otherfemale conspecifics has been well-documented in recent studies employinginfra-human mammals. See McClintock, M. K. 1983, supra., McClintock, M.K. 1984, Izard, M. K. 1983, supra., and Izard, M. K. and Vandenbergh, J.G. 1982, supra. For example, the estrous cycle of rats can bemanipulated through the use of odors collected from females duringspecific phases of the estrous cycle. In several rodent species,exposure to the odors of urine or cage bedding or males can promoteestrous or stimulate ovulation. In some infra-human primates, males caninfluence cycle length. Female baboons denied a mating showedsignificantly longer cycles and rhesus monkeys showed a summeramenorrhea two to four months after a male decrease in sexual potency.

Notwithstanding what is known on this topic, the problems of unwantedfertility and unwanted infertility remain among the most important ofhuman concerns. Since a reasonable percentage of infertility may stemfrom conditions reflected in menstrual cycles of aberrant lengths,methods for adjusting the lengths of those menstrual cycles have a goodlikelihood of creating presumptively fertile 29.5±3 days cycles.Similarly, for those women choosing to avoid pregnancy, the provision ofa physiologically compatible method for stimulating infertile-typecycles would be of utmost utility. Finally, improved methods forregulating the timing of the menstrual cycle could improve thepredictability of the occurrence of the fertile period and/ormenstruation and expand the range of therapeutic possibilities foraddressing both fertility and infertility problems.

SUMMARY OF THE INVENTION

A novel method of determining the onset of the fertile period of a humanfemale, and of predicting the time of ovulation in that female, isprovided which comprises the step of monitoring the axillary secretionsof that female during the course of her menstrual cycle to determinevariations in the concentration of androstenol or dehydroepiandrosteronesulfate. First significant increases in the concentration of either ofthese compounds following the cessation of menses is indicative of theonset of the fertile period, and is predictive of ovulation in five toseven days.

In accordance with the preferred embodiment of the present invention,samples of axillary secretions of the subject female are collectedperiodically during her menstrual cycle beginning at the cessation ofmenses. This monitoring comprises determining the concentrations ofandrostenol or dehydroepiandrosterone sulfate in said samples, for thepurpose of determining at least a four fold, preferably at least a tenfold increase in the concentration of either of these compounds ascompared to their base levels immediately following menses. In eachcase, a follicular phase peak is indicative of the onset of the fertileperiod.

In accordance with the preferred embodiment method, indicator means isprovided for qualitatively and quantitatively responding to variationsin the concentration of androstenol or androsterone. Collected samplesof the axillary secretions may be tested by the indicator means, whichmay be a gas chromatograph/mass spectrometer or a radioimmunoassay (RIA)test, to determine the concentration of the subject compound. Althoughdaily collection is preferred, it may be possible to obtain indicativeresults by conducting periodic collections as infrequently as once everyfour days during the follicular phase of the menstrual cycle. However,the accuracy of the prediction of the onset of the fertile period issomewhat dependent upon the periodicity of the sampling procedure.

The present invention further provides improved birth control methodswherein the aforementioned procedures are combined with knowncontraceptive methods to facilitate their use beginning at the onset ofthe fertile period until at least about one day following the expectedtime of ovulation. In accordance with this birth control method, anadditional step of determining the presumptive time of ovulation may beemployed. This additional method may be clinical method, such as theinterpretation of a basal body temperature chart, or a chemical method,such as the determination of a peak in the concentration of a compoundwhich is indicative of the time of ovulation. A peak in the circulatingblood levels of luteinizing hormone, or in the concentration ofdodecanol in the female is saliva, or of certain volatile sulfurcontaining compounds in mouth air, or in the concentration of lacticacid, acetic acid, or urea found in that woman's vaginal secretions maybe used as a presumptive ovulation indicator.

The reliability of such a birth control method may be further improvedby improving the regularity of a woman's menstrual cycle. For example,the menstrual cycle of the human female may be adjusted by exposing thenasal region of that female to at least a portion of the axillarysecretions of a human donor, as described in connection with theaforementioned related patent applications, which have been incorporatedherein by reference. Such axillary secretions collected from one or morehuman males will, for example, tend to induce regular menstrual cycleshaving lengths of about 29.5±3 days. Alternatively, axillary secretionsfrom at least one other human female selected in accordance with herknown menstrual cycle history may act to adjust or disrupt the expectedmenstrual cycle of that female. In the former instance, the regularityof the induced cycle will improve the reliability of fertile period andovulation indicators, whereas in the latter the disrupted menstrualcycle will itself tend to prevent pregnancy.

Accordingly, a primary object of the present invention is the provisionof improved methods for determining the onset of the fertile period of ahuman female.

A further object of the present invention is the provision of a novelmethod for predicting the presumptive time of ovulation of that humanfemale.

A further object of the present invention is the provision of animproved birth control method.

Still a further aim of the present invention is the provision of amethod of improving the reliability of existing birth control methodswhich rely upon the sporadic use of contraceptive methods, materials ordevices during a portion of the menstrual cycle of the human female.

These and other objects of the present invention will become apparentfrom the following, more detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the concentrations in nanograms per milliliter ofaxillary extract dehydroepiandrosterone. sulphate and androsteronesulphate for samples of axillary extracts pooled from five women donors,as determined over the course of the menstrual cycle;

FIG. 2 is a graph of the concentration in nanograms per milliliter, ofandrostenol for samples of axillary extracts pooled from five womendonors, as determined over the course of the menstrual cycle.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is predicated upon the finding that androstenol,and/or dehydroepiandrosterone sulfate exhibit distinctive cyclingbehaviors during the course of a woman's menstrual cycle, and that bymonitoring the concentrations of either or both of these compounds, itis possible to determine the onset of the fertile period of that humanfemale and/or to predict by five to seven days the time of ovulation.

Support for the above-stated conclusions comes from a study in whichaxillary secretions were collected from five female volunteer donorsrecruited from amongst co-workers and members of the community. All hadthe following characteristics: they were engaged in a heterosexualrelationship; they had large numbers of lipophilic diptheroids in theiraxillary region, and for the duration of the experiment they did not usedeodorant, deodorant soaps or perfumes in the axillary region; and theywashed each morning with only Ivory soap.

Secretions were collected on 4"×4" cotton pads which had previously beenextracted, autoclaved, dried and wrapped in solvent extracted aluminumfoil. Each donor wore a pad in each axilla three times a week during aconvenient six-to-nine hour periods. After removal, pads wereimmediately frozen in acid/solvent cleaned glass jars and stored at -60°C. until extraction, approximately one year later. Each donor collectedsecretions for three complete menstrual cycles. However, the axillarypads included in the preparation of the subject samples cam from onlyfive of the fifteen donated cycles. These cycles were selected becausethey met the following criteria: each was 29.5±2 days in length; thebasal body temperature charts were clearly biphasic and presumablyovulatory with thermal rises of twelve or more days, and menstruationoccurred within seven days of a full moon. To prepare the stimuli, padswere groups into three-day segments: e.g., all pads from cycle days 1-3were combined and called "Combined Donor Day 1" (the first day ofmenstration equals day 1 of the menstrual cycle), all pads in days 4-6were combined and called "Combined Donor Day 4", all pads in days 7-9were combined to form "Combined Donor Day 7", and so on. In thisfashion, ten separate extracts were prepared, each containing odors fromdifferent portions of the menstrual cycle to form the "Donor Cycle" ofcombined donor days 1, 4, 7, 10, 13, 16, 19, 22, 25 and 28. All padsfrom each three-day group were placed in a glass column and allowed tosoak in doubly distilled ethanol for one hour at room temperature.Fifteen mls of ethanol were used for each pad in the column. After onehour the ethanol extracted materials were allowed to run out the bottomof the column through a PTFE (Teflon) stop cock as the pads weresqueezed with a PTFE (Teflon) disc. Approximately two-thirds of allethanol put on the pads was recovered. The ethanol extracted sampleswere subsequently frozen at -60° C. until needed.

Subsequently, the steroid content of the extracts were examined and thetypes of odors present in the separated chromographic effluent weresurveyed. Quantitative gas chromotography/mass spectrometry usingportions of the individual combined donor days concentrated to a smallvolume (25-75 ml) was performed employing the "Target Compound Analysis"procedure which is part of the "INCOS" software on theFinnigan/MAT4510GC/MS System. Standard curves of the desired steroidsversus a standard (androstane) of constant concentration were firstcreated over a range of concentrations: dehydroepiandrosterone sulphate(DHAS: 1, 5 10 and 25 ng); androsterone sulphate (AS: 1, 5, 10 and 25ng); androstenol (0.25, 1, 5 and 10 ng); and androstenone (0.25, 1, 5and 10 ng).

Chromatography was carried out on a 25 m×0.25 mm (O.D.) fused silicacolumn coated with CP SIL-A (Chrompack, Inc., Bridgewater, N.J.), apermanently bonded methyl silica phase with a flow through the column ofapproximately 3.0 ml/min. All chromatography was done using thefollowing protocol: 100° C. for four minutes, then 4° C. per minute to300° C.

Analysis of the sulphates noted above are carried out by quantitation ofthe amount of their major pyrolysis product. See Labows, J.N., Preti, G.Hoelzle, E., Leyden, J. and Kligman, A. M. "Steroid Analysis of HumanApocrine Secretion", Steroids 34:249-258 (1979). As can be seen fromFIG. 1, the amount of DHAS is markedly elevated in the extract ofcombined donor day 7, representing combined days 7, 8 and 9. No suchelevation is seen for androsterone sulphate, however. The volatilesteroid androstenol also rises in concentration at this time, althoughthe levels seens for this compounds are far less that are seen for thesulphates (See FIG. 2). The levels of the compounds reported here areconsistent with previous reports examining the steroidal constituents ofthe axillae. See Labrow, J. N. 1979. "Human Odors--What Can They TellUs!", Perfumer and Flavorist 4:12-17 and Brooksbank, B. W. L., Brown, R.and Gustafsson, J. A. 1974, "The Detection of 5-androst-16-en-3-o1 inHuman Male Axillary Sweat", Experientia 30:864-865. In the presentstudy, androstenone amounts could not be determined because it elutesclosely to the pyrolysis product used for quantitation of androsteronesulfate. However, there appears to be far smaller amounts ofandrostenone as compared to the androstenol. Studies are currentlyunderway to reexamine freshly concentrated samples of thawed out extractwith a different temperature program to allow for resolution ofandrostenone from the As pyrolysis product so as to determine the levelsof androstenone.

It is presently believed that these are the first studies to investigatethe concentration changes of axillary secretion steroids across themenstrual cycle. These data indicate that a variation is present, andthat secretions from the mid-follicular phase are of particular interestbecause there appears to be a time of uniquely increased concentrationsof dehydroepiandrosterone sulfate and androstenol.

As seen from the above, this knowledge of the behavior of androstenoland dehydroepiandrosterone sulfate facilitates the provision of novelmethods for determining the onset of the fertile period of the humanfemale, and for predicting the time of ovulation of that female. Whileeach of the subject compounds exhibits a ten fold mid-follicular phasepeak, dehydroepiandrosterone sulfate exhibits more than a fifty foldincrease over the base level detected immediately following cessation ofmenses.

The present invention also provides a novel family planning (birthcontrol or intercourse timing) method wherein the aforementioned changesin concentration of androstenol and/or dehydroepiandrosterone sulfatemay be monitored by beginning at the cessation of menses until a firstpeak in the concentration of at least one of said compounds indicatesthe onset of the fertile period. Beginning no later than two days afterthe detection of this peak, and preferably beginning immediately at thetime of detection of this peak, the woman should begin to avoid or seekexposure to fertilization in accordance with conceptual planning. Suchavoidance should continue until about one day following the expectedtime of ovulation. If preferred, methods for determining the time ofovulation may be employed, which include clinical methods such as thebasal body temperature method, or chemical methods, such as thedetermination of a peak in the concentration of at least one compoundwhich is indicative of the time of ovulation. Such compounds include apeak in the circulating blood levels of luteinizing hormone, a peak inthe concentration of dodecanol in saliva, a peak in certain volatilesulfur containing compounds in mouth air, a peak in the concentration oflactic acid, acetic acid and urea found in vaginal secretions, or peaksin other compounds which are known to be indicative of ovulation.

By using the birth control method of the present invention,contraceptive methods, such as abstinence; contraceptive materials, suchas spermicidal foams; or contraceptive devices, such as condoms ordiaphragms, need be used for only a portion of the overall menstrualcycle. The reliability of these methods may be further improved byregulating the menstrual cycle of the human female by exposing her nasalregion to at least a portion of the axillary secretions of a humandonor. As described in the aforementioned related patent applicationssuch axillary secretions may include axillary secretions of at least onehuman male to thereby induce menstrual cycles having expected lengths of29.5 ±three days. Alternatively, axillary secretions of at least oneother human female exhibiting expected menstrual cycle lengths of 29.5±three days may be applied for a similar purpose, when applied insynchrony, or to disrupt the treated female's menstrual cycle todecrease the probability of pregnancy, when applied in an asynchronousmanner. Alternatively, the axillary secretions may be collected from ahuman female exhibiting a shortened luteal phase (less than eleven days)for the purpose of driving the treated female's menstrual cycle towardsa presumptively infertile length.

Accordingly, novel methods for improving the reliability of birthcontrol methods relying on the sporadic use of contraceptive methods,materials or devices during a portion of a menstrual cycle of a humanfemale are provided which comprise the step of exposing the nasal regionof the human female to an axillary secretion of another human for aduration and in an amount sufficient to adjust the length of themenstrual cycle of said human female to improve the predictability ofthe presumptive fertile period, thereby facilitating improvedreliability of the aforementioned contraceptive methods, materials ordevices.

What is claimed is:
 1. A birth control method comprising the stepsof:(a) determining the onset of the fertile period of a human female bymonitoring the variation in concentration in the axillary secretions ofsaid female of a compound selected from the group consisting ofandrostenol and a dehydroepiandrosterone, by beginning said monitoringat the cessation of menses until a first significant increase in aconcentration of at least one of said compounds indicates the onset ofthe fertile period; and (b) causing said human female to avoid exposureto fertilization beginning from about that time until at least about 1day following the expected time of ovulation.
 2. The method of claim 1wherein said dehydroepiandrosterone is dehydroepiandrosterone sulfate.3. The method of claim 1 wherein said monitored compound isandrosterone.
 4. The method of claim 1 wherein said monitored compoundis dehydroepiandrosterone.
 5. The method of claim 1 wherein said methodfurther comprises the step of determining the presumptive time ofovulation.
 6. The method of claim 5 wherein said step of determining thetime of ovulation is performed using the basal body temperature method.7. The method of claim 5 wherein said step of determining ovulation isperformed by determining a peak in the concentration of at least onecompound, the peak of which is indicative of the time of ovulation 8.The invention of claim 7 wherein said peak is a peak in luteinizinghormone.
 9. The method of claim 7 wherein said peak is a peak in theconcentration of dodecanol in said human female's saliva.
 10. The methodof claim 7 wherein said peak indicative of ovulation is a peak in theconcentration of a compound found in vaginal secretions, said compoundbeing selected from the group consisting of lactic acid, acetic acid,and urea.
 11. The method of claim 7 wherein said peak is a peak of avolatile sulfur compound present in mouth air.